Endotracheal intubation results in acute tracheal damage induced by mtDNA/TLR9/NF‐κB activity

Abstract Tracheitis secondary to placement of an endotracheal tube (ETT) is characterized by neutrophil accumulation in the tracheal lumen, which is generally associated with epithelial damage. Mitochondrial DNA (mtDNA), has been implicated in systemic inflammation and organ dysfunction following trauma; however, less is known about the effects of a foreign body on local trauma and tissue damage. We hypothesized that tracheal damage secondary to the ETT will result in local release of mtDNA at sufficient levels to induce TLR9 and NF‐κB activation. In a swine model we compared the differences between uncoated, and chloroquine (CQ) and N‐acetylcysteine (NAC) coated ETTs as measured by tracheal lavage fluids (TLF) over a period of 6 h. The swine model allowed us to recreate human conditions. ETT presence was characterized by neutrophil activation, necrosis, and release of proinflammatory cytokines mediated by TLR9/NF‐κB induction. Amelioration of the tracheal damage was observed in the CQ and NAC coated ETT group as shown in tracheal tissue specimens and TLF. The role of TLR9/NF‐κB dependent activity was confirmed by HEK‐Blue hTLR9 reporter cell line analysis after coincubation with TLF specimens with predetermined concentrations of NAC or CQ alone or TLR9 inhibitory oligodeoxynucleotide (iODN). These findings indicate that therapeutic interventions aimed at preventing mtDNA/TLR9/NF‐κB activity may have benefits in prevention of acute tracheal damage.


INTRODUCTION
Placement of an endotracheal tube (ETT) is an essential procedure performed during different aspects of medical care. The tracheal mucosa is exposed daily to a myriad of endogenous and exogenous agents that are recognized and controlled by the innate immune system, in particular, by neutrophils. Placement of an ETT is a procedure capable of inducing various degrees of tracheal tissue damage, 1-3 pain, 4 tracheitis, and possibly pneumonia. 5 Although studies have shown the effects of airway pathogens on local and systemic inflammation, 6 the early events that lead to tracheal neutrophil activation in the absence of infection have not been elucidated. Neutrophil luminal migration implies a barrier disruption of the epithelial layers that is suggestive recognition receptors (PRR), 18,19 among them TLRs 20 capable of recognizing antigens originated during sterile and nonsterile cellular injury. Notably, injury induces release of host cellular molecules known as damage associated molecular patterns (DAMPs) 21 that are readily recognized by TLRs. It is known, that the respiratory system expresses multiple TLRs including TLR2, TLR4, and TLR9 on cells lining the airway. 22 [25][26][27][28] is a danger signal that may induce a potent immune mediated inflammatory response following engagement of TLR9. Organ dysfunction secondary to mtDNA release and induction of sterile injury has been demonstrated during cardiac dysfunction, 28 and tracheal 17 and lung injury. 25 Previously, in human subjects who received an ETT we documented higher concentrations of mtDNA and TLR9 in aseptic tracheal lavage fluids (TLF) of patients suffering from sore throat as compared to those without sore throat. 17 Signaling of TLR9 is known to occur via MyD88 resulting in transcription of several hundred genes mediated by NF-B. 29,30 Activation of NF-B has been observed in studies of systemic inflammatory response syndrome (SIRS), 31 bowel inflammation, 32 and arthritis, 33 as well as in our human tracheal study, 17 resulting in gene expression of proinflammatory cytokines.
There is no effective method to ameliorate tracheal damage induced by mtDNA release secondary to a foreign body. In this study, we hypothesize that tracheal tissue damage induced by an uncoated ETT would promote neutrophil activation, migration, and necrosis via TLR9/NF-B activation. We additionally aimed to determine the effects of medications chloroquine (CQ; anti-inflammatory) and Nacetylcysteine (NAC; ROS scavenger) as a coating for ETT tubes to limit cellular injury.

Study design
After approval by the Animal Studies Committee at Washington University in St. Louis, and in accordance to the guidance stipulated by the Animal Welfare Act and the Association for the Assessment and Accreditation of Laboratory Animal Care (AAALAC), we conducted a prospective study in swine to evaluate the impact of an ETT on tracheal tissue and neutrophil activation during 6 h of exposure. We randomized the swine to two groups (5 animals each), one uncoated and the other CQ/NAC coated. We utilized a 7 mm ID Mallinckrodt TM TaperGuard Evac ETT that was dipped in a mixture of CQ/NAC in 20% Polyvinylpyrrolidone (PVP)/10% ethanol for 15 min and dried in a sterile incubator at 37 • C +5% CO2. This coating and drying cycle was repeated two more times before the coated devices were returned to their original sterile packaging. Identical ETTs without coating were utilized in the other group of animals.
Ten healthy female pigs were anesthetized with 1 to 2 mg/kg of tiletamine, ketamine, xylazine (TKX) prior to intubation, and anesthesia was maintained with 1-3% isoflurane. ETT balloons were inflated with 10 mL of air titrated to cuff leaks at 20 cm/H 2 O pressure. Tracheal lavages were done at 0 (5-10 min after intubation), 3 and 6 h, with 10 ml of sterile saline solution using a push and suction technique with a wall mounted device set up at low suction pressures. Blood was also drawn from a peripheral vein at 0, 3, and 6 h. After ETT removal, pigs were euthanized and biopsies of the trachea at the point of contact were collected and preserved in formalin.

Neutrophil phenotypes and respiratory burst assessment
Cell pellets from the TLF specimens after centrifugation were manually broken apart by pipetting, and neutrophils were isolated by magnetic negative separation using EasySep TM Neutrophil Enrichment Kits (Stem Cell Technologies, Tukwila, WA, U.S.) ( Fig. 2A). Isolated neutrophils were stained with fluorochrome-conjugated antibodies for CD11a, CD11b, CD16, CD18, CD54, and CD62L for assessing activity and adhesion (Fig. 2C). 7-Aminoactinomycin D was used to assess cellular necrosis and Annexin V as a viability marker (Fig. 2B). Neutrophil respiratory burst was characterized by incubating cells for 10 min at 37 • C with 10 ng/mL phorbol 12-myristate 13-acetate followed by 10 s with 20 M dihydrorhodamine 123 (DHR123) (Fig. 6A). Neutrophil phenotypes and ROS were characterized by FACS and analyzed with FlowJo R X software (FlowJo, LLC). Total RNA was isolated from the TLF neutrophils from both groups via TRIzol R (Thermo Fisher Scientific, Carlsbad, CA, U.S.).

Mitochondria DNA quantification
TLF obtained from each time point were centrifuged, and supernatants were collected to quantitate mtDNA by quantitative polymerase chain reaction (qPCR) for swine cytochrome b (Mt-cyb) compared against a known concentration of mtDNA (Fig. 3). RT-PCR using primer/probe pairs specific to porcine cytochrome b (Mt-cyb), which is encoded in mtDNA but not encoded in genomic DNA made a distinction between mtDNA and total DNA. We compared the Mt-cyb values to known concentration of Mt-cyb DNA template provided by the primer manufacturer (qSscCEP 0027819, catalog numbers for primer 12001961, and template 10047280, Bio-Rad Laboratories, Inc., Hercules, CA, U.S.).

TLR9 expression in TLF neutrophils
Comparative analysis for TLR9 expression of TLF neutrophils (Fig. 3B) was done using mRNA from blood neutrophils as a baseline control instead of using a separate reference gene. Quantification was done using a qPCR cycle threshold (Ct) method of relative quantification in which concentrations of mRNA from all samples were adjusted in reference to each other as evaluated with a nanodrop 2000, and as described elsewhere. 34 We used Taqman TLR9 primer from Thermo Fisher Scientific with KAPA Probe Fast Master Mix.

Inflammatory markers in TLF
Extracellular concentrations of the inflammatory markers IL-1 , IL-6, IL-8, IL-10, and TNF-were determined by sandwich ELISAs

Tracheal histology
Tracheal tissue biopsies at the point of contact with the ETTs were taken from both uncoated and CQ/NAC coated swine groups and fixed in 10% formaldehyde for 48 h, washed and dehydrated in ethanol, and paraffin-embedded. Samples were then cut and stained with H&E. A veterinary pathologist blinded to the research conditions scored the histological specimens of at least 4 subjects for evidence of epithelial tissue injury/integrity, inflammation, and cellularity using a numerical scoring guideline scaled from 0 to 4, with 0 corresponding to normal or minimally injured tissue and no inflammatory cells, whereas 4 represented extensive tissue damage and elevated inflammatory cell infiltration (Table 1). Typically a swine trachea unexposed to an ETT has low cellularity (Supplemental Table 1).

Statistics
Concentrations of mtDNA were compared with Kruskal-Wallis and post hoc Dunn's tests, whereas mean fluorescence intensity (MFI) from FACS analysis, ELISA, and NE activity data were compared against inactive peripheral blood neutrophils with Wilcoxon Mann-Whitney U tests. All histology data was compared by a Student's t test. All data were analyzed using SPSS v.17.0 (SPSS, Inc., Chicago, IL, USA). Histology of swine tracheal tissues exposed to uncoated and CQ/NAC coated ETT. Scoring table for inflammatory cells represented by neutrophils, mononuclear cells and fraction of basal membrane that is covered by epithelial tissue. Lower scores represent better tissue preservation and higher scores correspond to tissue damage of various degrees. Tissues scored for pathology and infiltration of inflammatory cells. a P < 0.05. b P < 0.01 by Student's t test, considered significant.

Tracheal epithelium is disrupted by uncoated ETT
Previously, we have shown that surrogate markers of tissue injury were present in TLF obtained from human and swine subjects exposed to an uncoated ETT. 17 In the present study we aimed to analyze the impact of ETT exposure at 6 h. We compared the effects of an uncoated ETT against a CQ/NAC coated ETT, utilizing collected TLF samples and specimens from local tissues. Here we demonstrate in the H&E sections that in contrast to the normal tracheal tissue (Fig. 1A), placement of an ETT results in tracheal damage at the point of contact (Fig. 1B control). In contrast, tracheal tissue exposed to CQ/NAC coated ETT showed less tracheal epithelium damage ( Fig. 1C; arrows). Furthermore, in Table 1 analysis of the histopathology shows that tracheal tissues with inflammation and higher cellularity in the uncoated ETT group (13.25), whereas the CQ/NAC coated group was significantly decreased (12.25, *P < 0.05). Tracheal tissue neutrophils were significantly decreased in the coated group (**P < 0.01). It is noteworthy that normal tracheal tissues have low cellularity prior to ETT exposure ( Fig. 1A and Supplemental Table 2). However, following ETT exposure, epithelial disruption, and high cellularity are evident as indicated in

Luminal neutrophils and phenotypes associated with ETT induced damage
Acute innate immune response in a variety of tissues is mediated predominantly by neutrophils. 17,[35][36][37] In order to understand the neutrophil response to the ETT, we analyzed TLF from subjects exposed to an ETT, and compared them to CQ/NAC coated ETT subjects. The uncoated ETT group was the control group because it reflects the current clinical standard of care. Therefore, we focus on the tracheal luminal neutrophils following migration after exposure to the ETT, and F I G U R E 1 Comparative Tracheal histology analyzed at the point of contact between the endotracheal tube and tracheal tissue. Tissues representative of tracheal tissues exposed to direct contact with uncoated and CQ/NAC coated endotracheal tubes (ETTs). 1A) Trachea from a normal swine with no ETT at 100× and 200× with arrows pointing to intact epithelium and ciliation. 1B) Epithelial disruption with multicellular migration and magnified at 100× and 200× with arrows to highlight the extensive lack of ciliation and tissue damage in the areas of contact with an uncoated ETT. 1C) Better preserved tracheal tissue with significant reduction of cellular infiltrate and magnified at 200× highlights presence of an intact epithelium and ciliated structures with arrows following exposure to a CQ/NAC coated ETT as indicated by Fig

Mitochondrial DNA/TLR9/NF-B pathway activation during injury
The release of mtDNA is a byproduct of cellular damage that is well established; thus we aimed to quantify mtDNA in the TLF in our study (6 h). As indicated in Fig. 3A we were able to detect under 2 g of mtDNA from both study groups. Consistent with acute injury, we detected statistically significant high amounts of mtDNA in TLF of the uncoated specimens at 6 h, which contrast the observed levels in the CQ/NAC coated group (Fig. 3A). These data are consistent with our hypothesis comparing coated and uncoated ETT at 6 h. Subsequently, we used TLR9, a known receptor for mtDNA, as an indirect marker to assess the effect of increased mtDNA (Fig. 3B). As expected, we detected increased TLR9 transcript in the TLF from the uncoated group consistent with the increased mtDNA in this group (Fig. 3).

Increased IL-8 is associated with acute injury in uncoated ETT
Because neutrophilia is a hallmark of tracheal epithelial damage, we

Impact of coated ETT on ROS and elastase activity
Excessive levels of ROS and elastase have significant impact on cellular activity and tissue damage. As indicated in Fig. 2A, the numbers of neutrophils in the TLF was persistently elevated in the uncoated group, and thus the damage induced by intubation is associated with ROS and elastase production (Fig. 6A, B). The apparent reduction of ROS in the

DISCUSSION
We present a model of tracheal luminal damage whereby placement and duration of an ETT exposure results in acute epithelial damage, neutrophilia, and DAMP release. The acute damage is characterized by localized release of mtDNA from epithelium and activated neutrophils. is promoted by indwelling ETTs. [1][2][3][4] The tracheal tissue is constantly exposed to antigens from external and internal sources and relies in an efficient mobilization of the innate immune system in particular neutrophil cells to provide a timely response. [35][36][37][38] Placement of an ETT results in trauma and provokes an exuberant response from the innate immune system increasing susceptibility to pain, 17,39 infection, 40 and tissue damage. 17,41 The end result of this activation is variable, ranging from sore throat (humans) to tracheitis [15][16][17]40 and tracheal stenosis. 42 Our study provides evidence of histological features representing tissue damage associated with the ETT, heralded by neutrophil luminal migration, extravasation, and accumulation in tracheal tissues.
In contrast, tracheal tissue histology following exposure to the anti- CD11b expressing myeloid cells such as macrophages. 50,51 Although the life cycle of a neutrophil is short in the circulation, following migration and activation, apoptosis can be delayed as they migrate and enter intraluminal spaces. 38,50 Necrosis is a process known to result in cell lysis after exposure to highly toxic substances that culminate in release of DAMPs, among them mtDNA which result in induction of an immune-inflammatory process. [52][53][54] Here, we demonstrated that luminal neutrophil cells not only accumulate in the tracheal lumen but also undergo significant necrosis and likely represent a significant source of mtDNA during tracheal injury. Although we did not attempt to directly block TLR9 in vivo, we observed that exposure to an ETT results in necrotic bias of neutrophils under conditions of elevated mtDNA/TLR9 presence ( Fig. 2A,B). As a result of cellular necrosis several local changes may result in tissue damage including release of mtDNA, which in turn activates TLR9 and may promote further release of ROS and tissue damage. ROS is essential for multiple homeostatic cellular functions, but uncontrolled activity results in cell injury [55][56][57]  and TNF-were documented in a study of patients with SIRS, whereas IL-6 and IL-10 were correlated with poor prognosis. 77 Interestingly, IL-10 presence has been documented in autoimmune disease processes in which a deficiency may result in intestinal inflammation. 78 IL-1 is also implicated in inflammasome activity in a caspase-1 dependent manner illustrating the complexity of the local tissue homeostasis.
Similarly, we observed that neutrophils obtained from TLF of swine receiving uncoated ETTs had a significant level of proinflammatory cytokine secretion. Up-regulation of NF-B mediated cytokine gene expression was also demonstrated in our study, an effect that was ameliorated after the use of predetermined amounts of CQ/NAC in the coated ETTs.
Some outstanding questions remain regarding neutrophil activation mediated by aseptic molecules. We need to consider potential cytosolic mtDNA inflammatory targets such as inflammasomes 79